Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.
The early development of the vertebrate nervous system is controlled by local cell interactions that determine the identity of specific neural cell types and the pathways of growing axons. One of the first cell types to differentiate within the embryonic nervous system is the floor plate, a small group of epithelial cells located at the ventral midline of the neural tube (Schoenwolf and Smith, 1990). The differentiation of the floor plate is induced by local, possibly contact-dependent signals from the notochord (FIG. 1) (van Straaten et al., 1988; Placzek et al., 1990c; Hatta et al., 1991). Signals that derive from the floor plate have been implicated in the control of cell identity in the neural tube and in the guidance of axons (FIG. 1) (Jessell and Dodd, 1991).
Evidence that the floor plate is a source of polarizing signals that control cell identity and pattern in the neural tube has come from experiments in chick embryos in which floor plate cells grafted next to the neural tube of host embryos give rise to additional ectopic motor neurons and to other ventral neuronal types defined by cell specific antigenic markers (Yamada et al., 1991; Placzek et al., 1991). Inversely, preventing floor plate differentiation by removing the notochord leads to the formation of a spinal cord that is devoid of motor neurons and other ventral neurons. These grafting experiments suggest that the floor plate has a central role in establishing the identity and pattern of neuronal cell types present in the ventral spinal cord. The floor plate also has limb polarizing activity when grafted into the chick wing bud, possibly through the release of morphogenically active retinoids (Wagner et al., 1990).
After the identity of spinal cord neurons has been established, the floor plate appears to provide both long-range and local guidance cues that promote the growth of axons to and across the ventral midline of the spinal cord. First, the floor plate secretes a diffusible chemoattractant which can orient the growth of axons of commissural neuron in vitro (FIG. 1) (Tessier-Lavigne et al., 1988; Placzek et al., 1990a; Tessier-Lavigne and Placzek, 1991) and may account for the homing of these axons to the floor plate in vitro (Weber, 1938; Placzek et al., 1990b; Bovolenta and Dodd, 1991; Yaginuma and Oppenheim, 1991). Second, the floor plate may contribute to the change in trajectory of commissural axons from the transverse to the longitudinal plane that occurs immediately after crossing the ventral midline (FIG. 1) (Holley and Silver, 1987; Dodd et al., 1988; Bovolenta and Dodd, 1990). In support of this proposal, genetic mutations in mice and zebrafish that result in the absence of the floor plate during embryonic development lead to errors in the pathfinding of commissural axons at the midline of the spinal cord (Bovolenta and Dodd, 1991; Bernhardt and Kuwada, 1990). Third, the floor plate may promote the fasciculation of commissural axons that occurs after they cross the midline of the spinal cord (Holley and Silver, 1987) by regulating the expression of glycoproteins of the immunoglobulin superfamily (Dodd et al., 1988; Schachner et al., 1990; Furley et al., 1990). The specialized role of the floor plate in vertebrate neural development has parallels in invertebrate organisms in that cells at the midline of the embryonic drosophila and C. elegans central nervous systems have been implicated in neural patterning and axon guidance (Klambt et al., 1991; Nambu et al., 1991; Hedgecock and Hall, 1990).
To identify molecules that may mediate the diverse functions of the floor plate during early neural development we have used subtractive hybridization techniques to isolate cDNA clones expressed selectively by the floor plate. We describe here the characterization of cDNA clones encoding a novel secreted protein, F-spondin, that is expressed at high levels by the rat floor plate during embryonic development. The predicted amino acid sequence of F-spondin reveals that the protein contains domains similar to those present in the thrombospondin and other proteins implicated in cell adhesion and neurite outgrowth. In vitro assays show that F-spondin promotes neural cell adhesion and neurite outgrowth suggesting that the secretion of this protein by the floor plate contributes to the growth and guidance of axons in the developing CNS.
The floor plate is a transient neural cell group implicated in the control of cell pattern and axonal growth in the developing vertebrate nervous system. To identify molecules that might mediate the functions of the floor plate we have used subtractive hybridization techniques to isolated and characterize floor plate-enriched cDNA clones. One such clone encodes a novel secreted protein, F-spondin, which is expressed selectively and at very high levels in the floor plate during early spinal cord development. The F-spondin gene necodes a protein of 90,000 molecular weight. The carboxyl terminal hal of the protein contains 6 repeats identified previously in thrombospondin and other proteins that have been implicated in cell adhesion and neurite outgrowth. F-spondin is expressed in the floor plate at the time that spinal axons first extend and at a lower levels in peripheral nerve. F-spondin is secreted from transfected cos cells and is also associated with the cell surface possibly by binding to the extracellular matrix. Recombinant F-spondin promotes the attachment of spinal cord and dorsal root ganglion cells and the outgrowth of neuritis form sensory neurons in vitro. These results suggest that F-spondin may contribute to the growth and guidance of axons in both the spinal cord and the peripheral nervous system.